C-terminal Elements Control Location, Activation Threshold, and p38 Docking of Ribosomal S6 Kinase B (RSKB)

Abstract

RSKB, a p90 ribosomal S6 protein kinase with two catalytic domains, is activated by p38-and extracellular signal-regulated kinase mitogen-activated protein kinase pathways. The sequences between the two catalytic domains and of the C-terminal extension contain elements that control RSKB activity. The C-terminal extension of RSKB presents a putative bipartite 713 KRX 14 KRRKQKLRS 737 nuclear location signal. The distinct cytoplasmic and nuclear locations of various C-terminal truncation mutants supported the hypothesis that the nuclear location signal was essential to direct RSKB to the nuclear compartment. The 725 APLA-KRRKQKLRS 737 sequence also was essential for the intermolecular association of RSKB with p38. The activation of RSKB through p38 could be dissociated from p38 docking, because RSKB truncated at Ser 681 strongly responded to p38 pathway activity. Interestingly, ⌬ 725-772 -RSKB was nearly nonresponsive to p38. Sequence alignment with the autoinhibitory C-terminal extension of Ca ؉2 /calmodulin-dependent protein kinase I predicted a conserved regulatory 708 AFN 710 motif. Alanine mutation of the key Phe 709 residue resulted in strongly elevated basal level RSKB activity. A regulatory role also was assigned to Thr 687 , which is located in a mitogen-activated protein kinase phosphorylation consensus site. These findings support that the RSKB C-terminal extension contains elements that control activation threshold, subcellular location, and p38 docking.The p90 ribosomal S6 protein kinases (RSKs) 1 are a family of Ser/Thr protein kinases composed of two catalytic domains, each with canonical ATP-binding site and activation loop sequences. In addition, RSKs contain a regulatory linker sequence connecting the two kinase domains and an extended C-terminal tail. RSKs comprise RSK1-RSK3, which are stimulated through the ERK pathway (1-6); the more recently identified mitogen-and stress-activated protein kinase type 1 and RSKB, which are activated by both the p38 and ERK pathways (7-9); and RSK4 (10). RSKs are involved in many diverse functions, such as regulation of glycogen metabolism by phosphorylating glycogen synthase kinase-3 and the G-subunit of protein phosphatase 1, and cell survival of cerebellar neurons through phosphorylation of BAD (reviewed in Refs. 11-13). RSKs function in the control of M phase entry of oocytes during meiosis and chromatin remodeling through histone H3 phosphorylation (14, 15). Furthermore, RSKs participate in the regulation of transcription factors and coregulators, such as CREB (3-5, 7, 8), CREB-binding protein and p300 (16), c-Fos (17), and estrogen receptor (18). Deficient mutants of RSK2 in man are linked to Coffin-Lowry syndrome, characterized by mental retardation and malformations (19). Deletion of RSK4 is common in patients with X-linked mental retardation (10). Interestingly, RSKB maps to the BBS1 locus (20), which is associated with Bardet-Biedl syndrome with manifestations reminiscent of Coffin-Lowry syndrome (21), and may be a candidate BBS gene.Many Ser/Thr-ki...

Identification of novel phosphorylation sites in MSK1 by precursor ion scanning MS

“…Mutation of Thr 700 to alanine slightly increased the basal activity of MSK1, while mutation to aspartic acid increased the basal activity still further. A similar result has been shown for mutagenesis of the equivalent site of MSK2 [30], although this study did not directly establish that this residue could be phosphorylated. Mutation of the equivalent site in MNK1 or MNK2 has also been reported to give similar results [31,32]; however, while this site is phosphorylated in vitro by ERK [33], it was unclear from these studies if this was an in vivo phosphorylation site.…”

“…Consistent with this, C-terminal truncation of MSK2 has been reported to increase its basal activity [30], although this truncation also deletes nuclear localization and MAPK docking sequences. Such a mechanism would explain the increased basal activity of the Thr 700 MSK1 mutations; however, it would not explain why Thr 581 phosphorylation is reduced in these mutants.…”

ERK and p38 MAPK-Activated Protein Kinases: a Family of Protein Kinases with Diverse Biological Functions

“…2). This docking sequence is necessary for ERK1/2 docking to RSKs (62,165,189) and MNK2B (170), ERK1/2 and p38 binding to MSK2 (207), as well as p38 docking to MK5 (179). It is likely that MAPK docking specificity arises from variations in the D domain sequence and from the potential involvement of other unidentified regions within the MKs.…”

Functional Characterization of Human RSK4, a New 90-kDa Ribosomal S6 Kinase, Reveals Constitutive Activation in Most Cell Types

“…Moreover Ser 742 appears to be phosphorylated by the activated NTK (28,36), which results in decreased affinity of RSK for ERK, serving as an intramolecular feedback inhibitory mechanism that operates in RSK1 and RSK2 but not in RSK3 (36). The activation mechanism of MSK is thought to be very similar to that of RSK except for two features (22,23,(37)(38)(39). First, the MAP kinase docking site can interact with both ERK and p38 MAP kinase, explaining why MSK is activated by two MAP kinase pathways.…”